Summary of Work: We have previously shown that estrogen-stimulated transcriptional activity of human lactoferrin gene in endometrium carcinoma cells, RL95-2, is mediated through a functional imperfect estrogen response element (iERE) present in the 5' flanking region of the gene. Upstream from the iERE, an extended steroid receptor half-site is protected by hERR a1 from DNase I digestion. From the electrophoresis mobility shift assay (EMSA), site-directed mutagenesis and DNA methylation interference analyses, we showed that binding of hERR a1 to the extended half-site of the steroid receptor enhances ER-mediated transcriptional activity of the human lactoferrin promoter. Mutation at the Gs in this region abolishes hERR a1 -DNA complex formation and reduces the estrogen responsiveness of the reporter plasmid containing the intact lactoferrin iERE. To examine the mechanism by which the hERR a1 and its DNA binding element enhance the estrogen response, we performed far-western and yeast two-hybrid analyses. The results showed that hERR1 interacted with estrogen receptor by direct protein-protein contact. Recently, we cloned and characterized the hERR a1 gene. By Fluorescent In-Situ Hybridization method (FISH), the hERRa gene was localized to the centromere region of chromosome 11q12. Partial sequencing, restriction mapping and PCR analysis revealed that the hERRa gene consists of seven exons and is over 20 kb in length. All intron/exon junctions follow the GT/AG rule. The highly conserved DNA binding domain of the hERRa1 is present in exon 2 and 3. Primer extension and RNase protection analyses reveal multiple transcription initiation sites in different human cell lines. The sequence immediately 5' of the transcriptional start sites of the hERRa1 message lacks a typical TATA box and CAAT box but is GC-rich and contains ten putative Sp1 binding sites. The region that contains the multiple Sp1 binding sites showed high levels of promoter activity when transiently transfected into the RL95-2 cells. We also isolated the cDNA of the mouse homologue of the hERRa and found that they share 97% homology in deduced amino acid sequence. Diethylstilbestrol (DES) stimulated the expression of ERR a mRNA in the uterus of 19-day-old mouse. We showed that DES but not progesterone or dexamethasone, enhanced the level of immunoreactive ERRa1 in the mouse uterus. These results demonstrated that hERRa modulates ER-stimulated activity by direct contact with the molecule and it is an estrogen responsive gene in the mouse uterus.